Clefts of the lip and palate are common human birth defects of multifactorial etiology with important dental, surgical, medical, speech, and behavioral implications. While the etiologies are complex, approximately 70% are non-syndromic and the subject of this proposal. In previous work, we have identified three candidate genes, TGFA, TGFB3 and MSX1, which contribute significantly to the causes of this disorder. In addition, environmental factors, particularly smoking, act as covariates with the candidate gene risk factors. Finally, using an affected pedigree member approach, we have mapped a locus for non-syndromic cleft lip and palate to 6p23. This previous work provides a frame onto which we can develop more detailed studies of the complex etiology of this disorder. In this renewal, we will carry out an expanded case ascertainment using surveillance systems in the U.S. and overseas, particularly in the Philippines under the auspices of Operation Smile. Collections of biologic specimens, family histories and exposure data will allow us to develop an even more powerful resource for high resolution genetic mapping using both linkage disequilibrium and affected pedigree member approaches, as well as for the incorporation of exposure data. Candidate gene studies will extend our molecular analysis to several new highly relevant candidate genes, including the retinoic acid receptor alpha and aryl hydrocarbon nucleotide translocator loci, both of which are strong candidates for gene- environment interactions. The affected pedigree member approach is now underway using a genome-wide search in a collaboration with Sue Malcolm, Mary Marazita and the Marshfield Clinic. We have in hand over 130 multiplex families from the Philippines and will build this collection to 500 in the course of this grant. APM and linkage-based approaches will provide the analytic engines to identify new loci. We will confirm our identification of the cleft lip and palate locus at 6p23 through high resolution genetic mapping and undertake an evaluation of relevant candidates already in this region, including AP2 and endothelin 1. Gene- environment interactions as joint risk factors for non-syndromic clefts will be extended through our larger population, looking at the independent contribution of parental environmental exposures and at joint gene- environment interactions. Long-term evaluations will include extensions of current gene expression work in mouse models, the incorporation of additional mouse knockouts into studies of gene-gene interactions and the examination of modifier effects. The outcome of this work, which has already successfully identified at least five important loci and/or genes for clefts in humans will be a more complete understanding of their developmental biology, with a goal toward improving treatment and prevention.